This invention relates to a method of sequencing oligosaccharides and, more particularly, to a method of oligosaccharide sequencing in which the components are determined essentially simultaneously.
Numerous analytical techniques for sequencing compounds are available which rely upon the use of well defined chemical or enzymatic reactions followed by the analysis of their products to identify the starting compound. For example, protein sequencing such as the Edman degradation has been widely used for many years for the direct determination of the primary structure of proteins and peptides [Edman, Acta Chem. Scand. 10, 761 (1956); Hunkapiller and Hood, Science 219, 650-659 (1983)]. The more recent introduction of rapid, simple methods of DNA sequencing also has become an important tool for biochemistry and molecular biology. The most widely used such DNA sequencing techniques are that of Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 74, 560-564 (1977), and Sanger et al., Proc. Natl. Acad. Sci USA 74, 5463-5467 (1977).
Methods have also been developed for determining the sequence of oligosaccharides such as that described by Kobata in "The Carbohydrates of Glycoproteins, Biology of Carbohydrates," (Ginsburg and Robins, Eds.), John Wiley and Sons, Vol. 2, pp. 87-162, 1984; Snider, Ibid., pp. 163-193, 1984. See also Harada et al., Anal. Biochem. 164, 374-381 (1987). Most proteins are glycoproteins which contain either O-glycosidically linked or N-glycosidically linked saccharides. These saccharides may vary from a single monosaccharide to highly branched structures containing over 30 monosaccharide residues. The determination of a monosaccharide sequence in such an oligosaccharide involves determining the order and branching pattern of the monosaccharide residues, the orientation of each glycosidic linkage (.alpha. or .beta.) and the linkage between the various monosaccharides. i.e. 1.fwdarw.3, 1.fwdarw.4, etc.
Most of the available analytical techniques for sequencing are sequential in nature, that is, a single reaction is performed and its products are analyzed, followed by a second reaction and a second analysis, performed either on the starting material or on the products of the first reaction. The sequential nature of these techniques can be illustrated by the following schematic outline: ##STR1##
These sequential techniques have the advantage of great flexibility and sensitivity. That is, each subsequent reaction can be selected on the basis of the previous results (flexibility), and the products of one reaction can be used as the starting point for the next (sensitivity). However, there also are disadvantages in these techniques in that the process can be slow, being a sequential technique, and difficult to automate unless the procedure is predefined, thereby resulting in loss of its flexibility.
Determination of the sequence and structure of oligosaccharides can be of significant importance in various fields, particularly in the medical and pharmaceutical fields. For example, the carbohydrate structure of a glycoprotein can have a significant effect upon its biological activity. That is, the oligosaccharides can affect the protein's antigenicity, stability, solubility and tertiary structure. The carbohydrate side-chains also can influence the protein's half-life and target it to receptors on the appropriate cells. The carbohydrate residues can affect both inter- and intracellular recognition. The sugar groups thus can control the relative effectiveness of a therapeutic protein when administered to a patient. These and other such functions of the carbohydrate moiety of glycoproteins are discussed, for example, by Delente, Trends in Biotech. 3(9), 218 (1985); van Brunt, Bio/Technology 4, 835-839 (1986); and Taunton-Rigby, Biotech USA 1988, Proc. Conf. San Francisco, Nov. 14-16, 1988, pp. 168-176.
It is also apparent that differences in the glycosylation pattern (i.e., particular structure at a specific site) on similar proteins or proteins with identical amino acid sequences can have profound effects on antigenicity, metabolism and other physiological properties. See, for example, the report on "The association of rheumatoid arthritis and osteoarthritis with changes in the glycosylation pattern of total serum" by Parekh et al., Nature 316, 452-457 (1985) and in U.S. Pat. No. 4,659,659.
The practical use of oligosaccharide sequencing also is illustrated with the medically important anti-thrombolytic glycoprotein known as tissue plasminogen activator (tPA) in U.S. Pat. No. 4,751,084. Improved methods of carrying out such oligosaccharide sequencing thus would have significant value in the medical and pharmaceutical fields and elsewhere.